In the cancer immunology and immunotherapy arena, we have solved a major paradox in the role of NKT cells in tumor immunosurveillance, as NKT cells have been reported by others to protect, and by us to suppress tumor immunosurveillance. We found that type I NKT cells (carrying the invariant Va14Ja18 T cell receptor) contribute to tumor immunity, whereas the type II NKT cells (with non-invariant receptor) are sufficient to suppress tumor immunosurveillance. We also showed that the type II NKT cell is the main cell suppressing immunosurveillance in 4 tumor models in which the classic CD4+CD25+ T regulatory cell does not play a role. Most recently, we discovered that protective type I NKT cells and suppressive type II NKT cells cross-regulate each other, defining a new immunoregulatory axis between this polar opposites. The balance between these may determine the immune response to tumors. We also found that blockade of the downstream mediator of this NKT regulatory pathway, TGF-beta, can synergize with an anti-cancer vaccine in mice, increasing the T cell response and more completely inhibiting growth of an established tumor (cervical cancer model) than the vaccine alone. The protection is CD8 T cell-dependent. We have a nearly completed phase I clinical trial of a human anti-TGF-beta monoclonal antibody in melanoma and renal cell cancer patients in the NIH Clinical Center as part of a CRADA with Genzyme Corporation. If successful, the intent is to use this agent in conjunction with a cancer vaccine. We also found that a recombinant adenovirus expressing Her-2 can prevent appearance of spontaneous autochthonous breast cancers in BALB-neuT Her-2 transgenic mice. The mechanism is antibody mediated and requires CD4 T cells only for help, and does not require CD8 cells. However, the protection is not FcR dependent, unlike that of Herceptin, so the antibody induced by the vaccine appears to block signal transduction by the oncoprotein, rather than induce killing by ADCC. We have further discovered that this vaccine can be used therapeutically to cure large (> 1 cm) established Her-2 positive tumors in mice as well as established lung metastases. This cancer vaccine may be more effective for breast cancer than the monoclonal Herceptin, because the patient would make her own antibodies and these would not be dependent on FcR-mediated mechanisms. We are working with a collaborator to develop an adenovirus expressing human Her-2 in preparation for a clinical trial in human breast cancer, and have had pre-pre-IND discussions with the FDA. We have mapped a new epitope presented by HLA-B7 from the PAX-FKHR fusion protein unique to and expressed by 85% of alveolar rhabdomyosarcoma patients. In addition, we have mapped HLA-A2-restricted epitopes from 4 new cancer antigens studied in collaboration with Ira Pastans lab, all expressed in prostate cancer but several also expressed in breast cancer and some other tumors. We have modified their sequences by epitope enhancement to make them more immunogenic, and have shown killing of human tumor cells by T cells specific for the first two of these. The first of these to be developed, TARP, is the subject of our planned clinical trial in prostate cancer patients that has been approved by the PRMC but has been delayed in obtaining an IND because of personnel turnover in the NIH Clinical Center Department of Transfusion Medicine that will prepare the dendritic cells for immunization. We anticipate that this trial will be opened within a few months. In the HIV and viral vaccine arena, first in studies also applicable to cancer vaccines, we have found that IL-15 expression by a vaccine will lead to induction of higher avidity cytotoxic T lymphocytes (CTL) that more effectively clear virus infections (or kill tumor cells). We also found that IL-15 will overcome the lack of CD4 T cell help in CD4 deficient animals, allowing induction of long-lived memory CTL that otherwise would not be induced without CD4 help. This may be a critical finding for therapeutic vaccines for HIV, for which it will be necessary to immunize HIV-infected or cancer patients with a deficiency of CD4 T cell help. We also found that the vaccinia-IL-15 vaccine was a more effective smallpox vaccine than the control vaccinia. Further, we found that the CD8 T cell avidity was a major factor in determining which epitopes of an antigen were immunodominant, more so than the level of expression of the epitope. We have also carried out epitope enhancement of HIV helper and CTL epitopes presented by human class II and class I HLA molecules, to make improved HIV vaccine constructs. Using one of these, we have made a vaccine to provide CTL-mediated counter selective pressure against the appearance of drug resistant mutants of HIV that escape from treatment with the anti-retroviral drug lamivudine (3TC). This approach is now being tested in a clinical trial carried out in collaboration with us by Bob Yarchoan in the HIV and AIDS Maligancy Branch of the CCR. We also showed that the TLR9 ligand CpG oligonucleotides could enhance the efficacy of a live viral vector vaccine, MVA, reducing the dose necessary to protect, and to induce protective CD8 CTL. Strikingly, the CpG oligos used with the MVA also allowed induction of CTL and protection in CD4 deficient mice. This may be another approach to a therapeutic vaccine for use in CD4 deficient patients infected with HIV. Further, we showed in mouse and macaque studies that clearance of virus from gut mucosal tissues, where HIV predominantly replicates, is dependent on having high avidity CTL in the local gut mucosa. Protection correlates with high avidity CTL, not low avidity CTL, in the gut mucosa, and not with CTL in the peripheral blood. Since the gut mucosa is a major reservoir for HIV replication, inducing mucosal high avidity CTL to eradicate this reservoir may be critical to keep HIV under control. We found in both mice and macaques that local mucosal immunization induces higher avidity CTL than immunization at more distant sites, and that avidity of CTL induced is correlated with the proximity to the site of vaccination, whether mucosal or systemic. We also recently showed proof of principle in macaques that it is possible for a mucosal AIDS vaccine to induce sufficient CTL in the mucosa to significantly delay dissemination of virus to the bloodstream and beyond. Although we did not completely prevent transmission, this delay indicates that a substantial fraction of the virus was eradicated at the initial mucosal site of infection, so that an improved version of this approach may be capable of eradicating the initial nidus of infection and thus aborting the AIDS infection before it becomes established. To improve this vaccine, we have been studying other TLR ligands as mucosal vaccine adjuvants, and have found a synergistic triple combination of TLR ligands that is more effective than any one (or pair) of these alone at inducing CTL responses. We also worked out the mechanism of synergy. We are now completing a macaque study using this synergistic combination of TLR ligands, or IL-15, or both, or neither, as vaccine adjuvants in combination with a peptide prime/recombinant MVA boost vaccine for SIV in macaques. If successful, this could pave the way to a vaccine to prevent mucosal transmission of HIV, the major natural route of HIV transmission